Compounds and methods for the prevention and treatment of tumor metastasis and tumorigenesis

ABSTRACT

The disclosure provides compounds for reducing the prevalence of the perinucleolar compartment in cells, for example, of formula (I), wherein R 1 , R 2 , R 3 , and R 4  are as defined herein, that are useful in treating a disease or disorder associated with increased prevalence of the perinucleolar compartment, such as cancer. Also disclosed is a composition containing a pharmaceutically acceptable carrier and at least one compound embodying the principles of the invention, and a method of treating or preventing cancer in a mammal.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.14/364,759, filed Jun. 12, 2014, which is a 371 of International PatentApplication No. PCT/US2012/070155, filed Dec. 17, 2012, which claims thebenefit of U.S. Provisional Patent Application No. 61/576,780, filedDec. 16, 2011, the disclosures of which are incorporated by reference intheir entirety herein.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant numbers R01GM078555 and R03 MH082371 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Metastasis is the cellular mechanism used by disease to spread from anorgan to another non-adjacent part of the organism. This process isparticularly important in the development of solid tumors and isresponsible for the majority of deaths associated with this disease. Itis well recognized in the field that treatment of a tumoral lesion has abetter prognosis if started in a pre-metastatic stage. In the lastdecade, although understanding of the underlying mechanisms involved inmetastasis has advanced, the therapeutic tools impacting specificallythe metastatic process are very limited.

SUMMARY

Inhibitors of the perinucleolar compartment (PNC), a subnuclear bodycharacterized by its location to the periphery of the nucleolus andwhich is associated with malignancy both in vitro and in vivo, aredisclosed as a solution to the unmet need for treating cancer,specifically the metastatic cancers. Compounds embodying aspects of theinvention disrupt the assembly of the PNC. Such disruption reduces(without overt cytotoxicity) the prevalence in cells of a multicomponentsubnuclear structure that is highly prevalent in metastatic tumors andfor which presence (of the structure) positively correlates withmetastatic capacity. In accordance with the invention, the presentdisclosure provides compositions comprising these compounds and methodsof using these compounds as therapeutic agents in the treatment ofcancer.

The disclosure provides a pharmaceutical composition comprising acompound or salt embodying the principles of the invention and apharmaceutically acceptable carrier.

The disclosure further provides a method for treating or preventingcancer in a mammal, comprising administering to a mammal in need thereofa compound embodying the principles of the invention or apharmaceutically acceptable salt thereof.

The disclosure additionally provides a method for disrupting a PNC in acell, comprising contacting the cell with a compound embodying theprinciples of the invention or a pharmaceutically acceptable saltthereof.

The disclosure further provides a method for reducing the prevalence ofperinucleolar compartment in a cell, comprising contacting the cell witha compound embodying the principles of the invention or apharmaceutically acceptable salt thereof.

The disclosure further provides a method for reducing ATP levelsproduced by metastatic cancer cells, in a mammal afflicted withmetastatic cancer, comprising administering to a mammal in need thereofa compound embodying the principles of the invention or apharmaceutically acceptable salt the

The disclosure additionally provides a method for reducing the colonyformation of cancer cells in a mammal, comprising administering to amammal in need thereof a compound embodying the principles of theinvention or a pharmaceutically acceptable salt thereof.

The disclosure additionally provides a method for reducing the migrationof cancer cells in a mammal, comprising administering to a mammal inneed thereof a compound embodying the principles of the invention or apharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 illustrates a histogram representing the number and size of PC3Msoft agar colonies after 14 days of treatment with a representativeembodiment of the invention at two different concentrations, as well asrepresentative images of colonies observed at these concentrations.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a compound of the formula:

wherein R¹ is selected from alkyl, hydroxyalkyl, thioalkyl, alkoxyalkyl,alkylthioalkyl, cycloalkyl, hydroxycycloalkyl, hydroxycycloalkylalkyl,thiocycloalkyl, alkoxycycloalkyl, alkylthiocycloalkyl,dialkylaminoalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,arylalkyl, arylalkylpiperidin-4-yl, arylpiperazinylalkyl, andheteroarylalkyl,

R² is aryl or heteroaryl,

R³ is selected from H, alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl,and heteroarylalkyl,

R⁴ is selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl,

or a pharmaceutically acceptable salt thereof,

wherein R¹, R², R³, and R⁴, other than H, are optionally substituted onthe aryl and/or alkyl portion with one or more substituents selectedfrom halo, alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl,alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino,aminosulfonyl, hydroxyl, perfluoroalkoxy, alkylenedioxy, andalkylcarbonyl,

with the proviso that when R² and R³ are both unsubstituted phenyl andR⁴ is unsubstituted benzyl, R¹ is not 3-hydroxypropyl.

In accordance with an embodiment, R² is phenyl, optionally substitutedwith one or more substituents selected from halo, alkyl, hydroxyalkyl,thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl,amino, alkylamino, dialkylamino, and alkylcarbonyl.

In accordance with certain embodiments, R² is phenyl.

In accordance with any of the above embodiments, R³ is phenyl,optionally substituted with one or more substituents selected from halo,alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl,alkylthiocarbonyl, amino, alkylamino, dialkylamino, and alkylcarbonyl.

In accordance with any of the above embodiments, R⁴ is benzyl, whereinthe phenyl ring is optionally substituted with one or more substituentsselected from alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl,alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino,aminosulfonyl, hydroxyl, perfluoroalkoxy, and alkylcarbonyl.

In accordance with any of the above embodiments, R⁴ is benzyl.

In accordance with any of the above embodiments, R¹ is a 5 or 6-memberedheterocyclyl group having at least one hetero atom selected from O, N,and S; a hydroxy C₁-C₇ cycloalkyl group; a hydroxy C₁-C₆ alkyl group; aN,N-di(C₁-C₆ alkyl)amino C₁-C₆ alkyl group; a C₁-C₆ alkoxy C₁-C₆ alkylgroup; a heteroaryl C₁-C₆ alkyl group; a heterocyclyl C₁-C₆ alkyl group;phenyl C₁-C₆ alkyl group wherein the phenyl ring is substituted with oneor more C₁-C₆ alkoxy groups; N-benzyl piperazinyl; N-phenylpiperazinylalkyl; a phenyl C₁-C₆ alkyl group where the alkyl issubstituted with a hydroxy group; or a 5 or 6 membered heteroarylaminoC₁-C₆ alkyl group wherein the heteroaryl group has at least one heteroatom selected from O, N, and S.

In accordance with certain preferred embodiments, R¹ is selected fromthe following:

In accordance with certain specific embodiments, R² is phenyl, R³ isphenyl, R⁴ is benzyl, and R¹ is selected from the following:

In accordance with certain embodiments, R⁴ is 4-methoxybenzyl.

In accordance with certain preferred embodiments, R¹ is selected fromthe following:

In accordance with certain specific embodiments, R² is phenyl, R³ isphenyl, R⁴ is 4-methoxybenzyl, and R¹ is selected from the following:

In accordance with any of the above embodiments, R⁴ is phenylethyl,wherein the phenyl ring is optionally substituted with one or moresubstituents selected from alkyl, hydroxyalkyl, alkoxy, andalkoxycarbonyl.

In accordance with certain embodiments, R⁴ is phenylethyl.

In accordance with certain preferred embodiments, R¹ is selected fromthe following:

In accordance with certain specific embodiments, R² is phenyl, R³ isphenyl, R⁴ is phenylethyl, and R¹ is selected from the following:

In accordance with certain embodiments, R⁴ is heteroaryl C₁-C₆ alkyl.

In accordance with certain embodiments, R⁴ is

In accordance with certain preferred embodiments, R¹ is selected fromthe following:

In accordance with certain specific embodiments, R² is phenyl. R³ isphenyl, R⁴ is

and R¹ is selected from the following:

In accordance with certain embodiments, R⁴ is selected from4-aminosulfonylbenzyl, 4-trifluoromethoxybenzyl, 4-methoxybenzyl, andcyclopropylmethyl.

In accordance with certain preferred embodiments, R¹ is selected fromthe following:

Referring now to terminology used generically herein, the term “alkyl”means a straight-chain or branched alkyl substituent containing from,for example, 1 to about 6 carbon atoms, preferably from 1 to about 4carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of suchsubstituents include methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.

The term “alkenyl,” as used herein, means a linear alkenyl substituentcontaining at least one carbon-carbon double bond and from, for example,about 2 to about 6 carbon atoms (branched alkenyls are about 3 to about6 carbons atoms), preferably from about 2 to about 5 carbon atoms(branched alkenyls are preferably from about 3 to about 5 carbon atoms),more preferably from about 3 to about 4 carbon atoms. Examples of suchsubstituents include vinyl, propenyl, isopropenyl, n-butenyl,sec-butenyl, isobutenyl, tert-butenyl, pentenyl, isopentenyl, hexenyl,and the like.

The term “alkynyl,” as used herein, means a linear alkynyl substituentcontaining at least one carbon-carbon triple bond and from, for example,2 to about 6 carbon atoms (branched alkynyls are about 3 to about 6carbons atoms), preferably from 2 to about 5 carbon atoms (branchedalkynyls are preferably from about 3 to about 5 carbon atoms), morepreferably from about 3 to about 4 carbon atoms. Examples of suchsubstituents include ethynyl, propynyl, isopropynyl, n-butynyl,sec-butynyl, isobutynyl, tert-butynyl, pentynyl, isopentynyl, hexynyl,and the like.

The term “cycloalkyl,” as used herein, means a cyclic alkyl substituentcontaining from, for example, about 3 to about 8 carbon atoms,preferably from about 4 to about 7 carbon atoms, and more preferablyfrom about 4 to about 6 carbon atoms. Examples of such substituentsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and the like. The cyclic alkyl groups may be unsubstitutedor further substituted with alkyl groups such as methyl groups, ethylgroups, and the like. The term “cycloalkylalkyl,” as used herein, refersto an alkyl group linked to a cycloalkyl group and further linked to amolecule via the alkyl group.

The term “heterocyclyl,” as used herein, refers to a monocyclic orbicyclic 5- or 6-membered ring system containing one or more heteroatomsselected from the group consisting of O, N, S, and combinations thereof.The heterocyclyl group can be any suitable heterocyclyl group and can bean aliphatic heterocyclyl group, an aromatic heterocyclyl group, or acombination thereof. The heterocyclyl group can be a monocyclicheterocyclyl group or a bicyclic heterocyclyl group. Suitable bicyclicheterocyclyl groups include monocylic heterocyclyl rings fused to aC₆-C₁₀ aryl ring. When the heterocyclyl group is a bicyclic heterocyclylgroup, both ring systems can be aliphatic or aromatic, or one ringsystem can be aromatic and the other ring system can be aliphatic as in,for example, dihydrobenzofuran. Non-limiting examples of suitablearomatic heterocyclyl groups include tetrahydrofuranyl,tetrahydropyranyl, tetrahydrothiopheneyl, pyrrolidinyl, piperidinyl, andmorpholinyl. Non-limiting examples of suitable aromatic heterocyclylgroups include furanyl; thiopheneyl; pyrrolyl; pyrazolyl; imidazolyl;1,2,3-triazolyl; 1,2,4-triazolyl; isoxazolyl; oxazolyl; isothiazolyl;thiazolyl; 1,3,4-oxadiazol-2-yl; 1,2,4-oxadiazol-2-yl;5-methyl-1,3,4-oxadiazole; 3-methyl-1,2,4-oxadiazole; pyridinyl;pyrimidinyl; pyrazinyl; triazinyl; benzofuranyl; benzothiopheneyl;indolyl; quinolinyl; isoquinolinyl; benzimidazolyl; benzoxazolinyl;benzothiazolinyl; and quinazolinyl. The heterocyclyl group is optionallysubstituted with 1, 2, 3, 4, or 5 substituents as recited herein such aswith alkyl groups such as methyl groups, ethyl groups, and the like, orwith aryl groups such as phenyl groups, naphthyl groups and the like,wherein the aryl groups can be further substituted with, for examplehalo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino,substituted amino, alkylcarbonyl, alkoxycarbonyl, arylcarbonyl,aryloxycarbonyl, thio, alkylthio, arylthio, and the like, wherein theoptional substituent can be present at any open position on theheterocyclyl group.

The term “heterocyclylalkyl,” as used herein, refers to an alkyl grouplinked to a heterocyclyl group and further linked to a molecule via thealkyl group.

The term “arylalkyl,” as used herein, refers to an alkyl group linked toa C₆-C₁₀ aryl ring and further linked to a molecule via the alkyl group.The term “alkylaryl,” as used herein, refers to a C₆-C₁₀ aryl ringlinked to an alkyl group and further linked to a molecule via the arylgroup.

The term “alkylcarbonyl,” as used herein, refers to an alkyl grouplinked to a carbonyl group and further linked to a molecule via thecarbonyl group, such as alkyl-C(═O)—. The term “alkoxycarbonyl,” as usedherein, refers to an alkoxy group linked to a carbonyl group and furtherlinked to a molecule via the carbonyl group, such as alkyl-O—C(═O)—.

Whenever a range of the number of atoms in a structure is indicated(such as a C₁-C₁₂, C₁-C₈, C₁-C₆, C₁-C₄, or C₂-C₁₂, C₂-C₈, C₂-C₆, C₂-C₄alkyl, alkenyl, alkynyl, etc.), it is specifically contemplated that anysub-range or individual number of carbon atoms falling within theindicated range also can be used. Thus, for instance, the recitation ofa range of 1-8 carbon atoms (such as C₁-C₈), 1-6 carbon atoms (such asC₁-C₆), 1-4 carbon atoms (such as C₁-C₄), 1-3 carbon atoms (such asC₁-C₃), or 2-8 carbon atoms (such as C₂-C₈) as used with respect to anychemical group (such as alkyl, alkylamino, etc.) referenced hereinencompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 carbon atoms, and combinations thereof, as appropriate, aswell as any sub-range thereof (such as 1-2 carbon atoms, 1-3 carbonatoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbonatoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms,3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms,3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbonatoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbonatoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11carbon atoms, and/or 4-12 carbon atoms, etc., as appropriate).Similarly, the recitation of a range of 6-10 carbon atoms (such as,C₆-C₁₀) as used with respect to any chemical group (such as, aryl)referenced herein encompasses and specifically describes 6, 7, 8, 9,and/or 10 carbon atoms, as appropriate, as well as any sub-range thereof(such as, 6-10 carbon atoms, 6-9 carbon atoms, 6-8 carbon atoms, 6-7carbon atoms, 7-10 carbon atoms, 7-9 carbon atoms, 7-8 carbon atoms,8-10 carbon atoms, and/or 8-9 carbon atoms, etc., as appropriate).

The term “halo” or “halogen,” as used herein, means a substituentselected from Group VIIA, such as, for example, fluorine, bromine,chlorine, and iodine.

The term “aryl” refers to an unsubstituted or substituted aromaticcarbocyclic substituent, as commonly understood in the art, and the term“C₆-C₁₀ aryl” includes phenyl and naphthyl. It is understood that theterm aryl applies to cyclic substituents that are planar and comprise4n+2 π electrons, according to Hückel's Rule.

The phrase “pharmaceutically acceptable salt” is intended to includenon-toxic salts synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two.Generally, non-aqueous media such as ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445, and Journal of PharmaceuticalScience, 66, 2-19 (1977).

Suitable bases include inorganic bases such as alkali and alkaline earthmetal bases, such as those containing metallic cations such as sodium,potassium, magnesium, calcium and the like. Non-limiting examples ofsuitable bases include sodium hydroxide, potassium hydroxide, sodiumcarbonate, and potassium carbonate. Suitable acids include inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalicacid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid, acetic acid, maleic acid, tartaric acid, fattyacids, long chain fatty acids, and the like. Preferred pharmaceuticallyacceptable salts of inventive compounds having an acidic moiety includesodium and potassium salts. Preferred pharmaceutically acceptable saltsof inventive compounds having a basic moiety (such as adimethylaminoalkyl group) include hydrochloride and hydrobromide salts.The compounds of the present invention containing an acidic or basicmoiety are useful in the form of the free base or acid or in the form ofa pharmaceutically acceptable salt thereof.

It should be recognized that the particular counterion forming a part ofany salt of this invention is usually not of a critical nature, so longas the salt as a whole is pharmacologically acceptable and as long asthe counterion does not contribute undesired qualities to the salt as awhole.

It is further understood that the above compounds and salts may formsolvates, or exist in a substantially uncomplexed form, such as theanhydrous form. As used herein, the term “solvate” refers to a molecularcomplex wherein the solvent molecule, such as the crystallizing solvent,is incorporated into the crystal lattice. When the solvent incorporatedin the solvate is water, the molecular complex is called a hydrate.Pharmaceutically acceptable solvates include hydrates, alcoholates suchas methanolates and ethanolates, acetonitrilates and the like. Thesecompounds can also exist in polymorphic forms.

In any of the above embodiments, the compound or salt of formula (I) canhave at least one asymmetric carbon atom. When the compound or salt hasat least one asymmetric carbon atom, the compound or salt can exist inthe racemic form, in the form of its pure optical isomers, or in theform of a mixture wherein one isomer is enriched relative to the other.In particular, in accordance with the present invention, when theinventive compounds have a single asymmetric carbon atom, the inventivecompounds may exist as racemates, that is as mixtures of equal amountsof optical isomers, that is equal amounts of two enantiomers, or in theform of a single enantiomer. As used herein, “single enantiomer” isintended to include a compound that comprises more than 50% of a singleenantiomer (that is enantiomeric excess up to 100% pure enantiomer).

When the compound or salt has more than one chiral center, the compoundor salt can therefore exist as a mixture of diastereomers or in the formof a single diastereomer. As used herein, “single diastereomer” isintended to mean a compound that comprises more than 50% of a singlediastereomer (that is diastereomeric excess to 100% pure diastereomer).

Synthetic Method

A general synthesis of embodiments of the compounds of the invention isdepicted in Scheme 1. The synthesis of the compound 104 commences withreaction of alpha hydroxyketone 100 with a primary amine in the presenceof catalytic zinc chloride to give the alpha aminoketone 101, which isnot isolated but reacts directly with malononitrile to give aminopyrrole102. Reaction of aminopyrrole 102 with triethyl orthoformate gives theimidate 103. Reaction of imidate 103 with primary amine R¹NH₂ in asolvent such as methanol provides final product 104.

The present invention is further directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one compound or salt described herein.

It is preferred that the pharmaceutically acceptable carrier be one thatis chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularcompound of the present invention chosen, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following formulations for oral, aerosol,nasal, pulmonary, parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginaladministration are merely exemplary and are in no way limiting.

The pharmaceutical composition can be administered parenterally, such asintravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration thatcomprise a solution or suspension of the inventive compound or saltdissolved or suspended in an acceptable carrier suitable for parenteraladministration, including aqueous and non-aqueous isotonic sterileinjection solutions.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See, such as Banker and Chalmers, eds., Pharmaceutics and PharmacyPractice, J. B. Lippincott Company, Philadelphia, pp. 238-250 (1982),and Toissel, ASHP Handbook on Injectable Drugs, 4th ed., pp. 622-630(1986). Such solutions can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The compound or salt of thepresent invention may be administered in a physiologically acceptablediluent in a pharmaceutical carrier, such as a sterile liquid or mixtureof liquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-beta-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations can contain preservatives and buffers. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range fromabout 5 to about 15% by weight. Suitable surfactants includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol. The parenteral formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

Topical formulations, including those that are useful for transdermaldrug release, are well-known to those of skill in the art and aresuitable in the context of the invention for application to skin.Topically applied compositions are generally in the form of liquids,creams, pastes, lotions and gels. Topical administration includesapplication to the oral mucosa, which includes the oral cavity, oralepithelium, palate, gingival, and the nasal mucosa. In some embodiments,the composition contains at least one active component and a suitablevehicle or carrier. It may also contain other components, such as ananti-irritant. The carrier can be a liquid, solid or semi-solid. Inembodiments, the composition is an aqueous solution. Alternatively, thecomposition can be a dispersion, emulsion, gel, lotion or cream vehiclefor the various components. In one embodiment, the primary vehicle iswater or a biocompatible solvent that is substantially neutral or thathas been rendered substantially neutral. The liquid vehicle can includeother materials, such as buffers, alcohols, glycerin, and mineral oilswith various emulsifiers or dispersing agents as known in the art toobtain the desired pH, consistency and viscosity. It is possible thatthe compositions can be produced as solids, such as powders or granules.The solids can be applied directly or dissolved in water or abiocompatible solvent prior to use to form a solution that issubstantially neutral or that has been rendered substantially neutraland that can then be applied to the target site. In embodiments of theinvention, the vehicle for topical application to the skin can includewater, buffered solutions, various alcohols, glycols such as glycerin,lipid materials such as fatty acids, mineral oils, phosphoglycerides,collagen, gelatin and silicone based materials.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as a therapeutically effective amount of the inventivecompound dissolved in diluents, such as water, saline, or orange juice,(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules,(c) powders, (d) suspensions in an appropriate liquid, and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant, suspending agent, or emulsifying agent. Capsuleforms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers,such as lactose, sucrose, calcium phosphate, and corn starch. Tabletforms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

The compound or salt of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. The compounds are preferably supplied infinely divided form along with a surfactant and propellant. Typicalpercentages of active compound are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such surfactants are theesters or partial esters of fatty acids containing from 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25%-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included asdesired, such as lecithin for intranasal delivery. These aerosolformulations can be placed into acceptable pressurized propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They alsomay be formulated as pharmaceuticals for non-pressured preparations,such as in a nebulizer or an atomizer. Such spray formulations may beused to spray mucosa.

Additionally, the compound or salt of the present invention may be madeinto suppositories by mixing with a variety of bases, such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, the compoundor salt of the present invention may be formulated as inclusioncomplexes, such as cyclodextrin inclusion complexes, or liposomes.Liposomes serve to target the compounds to a particular tissue, such aslymphoid tissue or cancerous hepatic cells. Liposomes can also be usedto increase the half-life of the inventive compound. Liposomes useful inthe present invention include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations, the active agent to be delivered isincorporated as part of a liposome, alone or in conjunction with asuitable chemotherapeutic agent. Thus, liposomes filled with a desiredinventive compound or salt thereof, can be directed to the site of aspecific tissue type, hepatic cells, for example, where the liposomesthen deliver the selected compositions. Liposomes for use in theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, for example, liposome size and stability of theliposomes in the blood stream. A variety of methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369. For targeting to the cells of aparticular tissue type, a ligand to be incorporated into the liposomecan include, for example, antibodies or fragments thereof specific forcell surface determinants of the targeted tissue type. A liposomesuspension containing a compound or salt of the present invention may beadministered intravenously, locally, topically, etc. in a dose thatvaries according to the mode of administration, the agent beingdelivered, and the stage of disease being treated.

The perinucleolar compartment (PNC) is a subnuclear body dynamicstructure, highly enriched in RNA-binding proteins and pol III RNA,which has been associated with malignancy both in vitro and in vivo. Inaddition, its presence positively correlates with metastatic capacity,making it a potential marker for cancer development and prognosis vivo(Pollock, C. et al., Cold Spring Harb Perspect Biol., 2010; 2(2), 1-10;Slusarczyk, A. et al., Cold Spring Harb Symp Quant Biol. 2010, 75,599-605).

Although the precise function of the PNC remains to be identified, PNCformation is closely associated with the metastatic phenotype. Notably,solid tumor cell lines seem to have a higher PNC population. A strikingobservation is the difference in PNC population between metastaticallytransformed cell lines and their parental counterparts. This observationparticularly holds for the PC-3M cell line that was created by removingand culturing a metastatic lesion after implantation of the humanprostrate tumor PC-3 cell line in nude mice. PNC prevalence (thepercentage of cells with at least one PNC) increases in parallel withdisease progression (staging and grading) for breast, ovarian, andcolorectal cancers and reaches near 100% in distant metastases. A highPNC prevalence in early stage of breast cancer associates with poorpatient outcomes (Kamath, R. V. et al., Cancer Res., 2005, 65(1),246-53). In addition, PNC prevalence directly correlates with the levelsof metastatic capacity in mouse metastasis models of human cancer cells.

PNCs are not associated with traits that are common in both cancer andnormal cells, such as proliferation, glycolysis, and differentiationstates. The selective association with metastasis makes PNC an ideal andsimple marker that reflects the complex trait of cellular malignancy.Thus, PNC reduction can be used as a phenotypic change to identify novelcompounds that may not directly target the PNC structure itself, butinduce desired changes that lead to the inhibition of cellularmalignancy.

Previous studies have shown that classical antitumoral agents, such astopoisomearse I and II inhibitors, DNA cross linkers, a subset ofnucleoside analogs, and methotrexate, cause reduction of PNC prevalence(Jin, Y. et al, Chem Biol., 2002, 9, 157-62; Norton, J. T. et al.,Anti-Cancer Drugs. 2008, 19(1), 23-36; Norton, J. T. et al., J. Biol.Chem. 2009, 284, 4090-4101). It has also been shown that the reductionof PNC by these agents is not a non-specific cytotoxic effect but aresult of inhibition of the molecular target. This is exemplified by DNAalkylators, microtubule disrupting drugs, hydoxyurea and some nucleosideanalogs which are cytotoxic agents that do not disrupt the PNC.Mechanistically it has also been suggested that those drugs which inducePNC reduction may be causing it via DNA damage. To that end, the DNAmight serve as a locus for the nucleation of the PNC and this notion isalso supported by the fact that the PNC is a heritable trait. Howeverall the agents that are known to reduce PNC have known mechanisms ofinducing cytotoxity, making it difficult to separate an anti-metasticeffect from cell death.

The invention further provides a method for treating cancer. The methodcomprises administering an effective amount of the compound of theinvention to an animal afflicted therewith. Preferably, the animal is amammal. More preferably, the mammal is a human.

The term “mammal” includes, but is not limited to, the order Rodentia,such as mice, and the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simioids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human. Furthermore, the subject can be theunborn offspring of any of the forgoing hosts, especially mammals (suchas, humans), in which case any screening of the subject or cells of thesubject, or administration of compounds to the subject or cells of thesubject, can be performed in utero.

In accordance with an embodiment, the invention provides a method oftreating or preventing cancer comprising administering to a patient inneed thereof a therapeutically effective amount of a compoundrepresented by Formula I, or a pharmaceutically acceptable salt thereof.The cancer can be any suitable cancer responsive to reduction of PNCprevalence, for example, cancers in which PNCs are prevalent.

In accordance with another embodiment, the invention provides a methodof treating cancer. The cancer can be any suitable cancer. Preferably,the cancer is a metastatic cancer. For example, the cancer may beadrenocortical carcinoma, AIDS-related lymphoma, AIDS-relatedmalignancies, anal cancer, cerebellar astrocytoma, extrahepatic bileduct cancer, bladder cancer, osteosarcoma/malignant fibroushistiocytoma, brain stem glioma, ependymoma, visual pathway andhypothalamic gliomas, breast cancer, bronchial adenomas/carcinoids,carcinoid tumors, gastrointestinal carcinoid tumors, carcinoma,adrenocortical, islet cell carcinoma, primary central nervous systemlymphoma, cerebellar astrocytoma, cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, clear cell sarcoma of tendonsheaths, colon cancer, colorectal cancer, cutaneous t-cell lymphoma,endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma/family of tumors, extracranial germ cell tumors, extragonadalgerm cell tumors, extrahepatic bile duct cancer, eye cancers, includingintraocular melanoma, and retinoblastoma, gallbladder cancer,gastrointestinal carcinoid tumor, ovarian germ cell tumor, gestationaltrophoblastic tumor, hairy cell leukemia, head and neck cancer,Hodgkin's disease, hypopharyngeal cancer, hypothalamic and visualpathway glioma, intraocular melanoma, Kaposi's sarcoma, laryngealcancer, acute lymphoblastic leukemia, acute myeloid leukemia, livercancer, non-small cell lung cancer, small cell lung cancer,non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, malignantmesothelioma, malignant thymoma, medulloblastoma, melanoma, intraocularmelanoma, merkel cell carcinoma, metastatic squamous neck cancer withoccult primary, multiple endocrine neoplasia syndrome, multiplemyeloma/plasma cell neoplasm, mycosis fungoides, myelodysplasticsyndrome, chronic myelogenous leukemia, myeloid leukemia, multiplemyeloma, myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavityand lip cancer, oropharyngeal cancer, osteosarcoma/malignant fibroushistiocytoma of bone, ovarian cancer, ovarian low malignant potentialtumor, pancreatic cancer, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor,pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell(kidney) cancer, transitional cell cancer (such as renal pelvis andureter), retinoblastoma, rhabdomyosarcoma, salivary gland cancer,malignant fibrous histiocytoma of bone, soft tissue sarcoma, sezarysyndrome, skin cancer, small intestine cancer, stomach (gastric) cancer,supratentorial primitive neuroectodermal and pineal tumors, cutaneousT-cell lymphoma, testicular cancer, malignant thymoma, thyroid cancer,gestational trophoblastic tumor, urethral cancer, uterine sarcoma,vaginal cancer, vulvar cancer, and Wilms' tumor.

In any of the embodiments of the invention, the cancer can be any cancerin any organ, for example, a cancer is selected from the groupconsisting of brain carcinoma, glioma, thyroid carcinoma, breastcarcinoma, small-cell lung carcinoma, non-small-cell carcinoma, gastriccarcinoma, colon carcinoma, gastrointestinal stromal carcinoma,pancreatic carcinoma, bile duct carcinoma, CNS carcinoma, ovariancarcinoma, endometrial carcinoma, prostate carcinoma, renal carcinoma,anaplastic large-cell lymphoma, leukemia, multiple myeloma,mesothelioma, and melanoma, and combinations thereof.

In an embodiment, the metastatic cancer is selected from the groupconsisting of breast cancer, ovarian cancer, colorectal cancer, braincancer, and prostate cancer.

In accordance with other embodiments, the invention provides a method ofpotentiating or enhancing anticancer activity of an anticancer agent,the method comprising coadministering to a patient in need thereof aneffective amount of an anticancer agent and a compound or salt of theinvention. The anticancer agent can be chosen from reversible DNAbinders, DNA alkylators, and DNA strand breakers.

Examples of suitable reversible DNA binders include topetecanhydrochloride, irinotecan (CPT11—Camptosar), rubitecan, exatecan,nalidixic acid, TAS-103, etoposide, acridines (such as amnsacrine,aminocrine), actinomycins (such as actinomycin D), anthracyclines (suchas doxorubicin, daunorubicin), benzophenainse, XR 11576/MLN 576,benzopyridoindoles, Mitoxantrone, AQ4, Etopside, Teniposide,(epipodophyllotoxins), and bisintercalating agents such as triostin Aand echinomycin.

Examples of suitable DNA alkylators include sulfur mustard, the nitrogenmustards (such as mechlorethamine), chlorambucil, melphalan,ethyleneimines (such as triethylenemelamine, carboquone, diaziquone),methyl methanesulfonate, busulfan, CC-1065, duocarmycins (such asduocarmycin A, duocarmycin SA), metabolically activated alkylatingagents such as nitrosoureas (such as carmustine, lomustine,(2-chloroethyl)nitrosoureas), triazne antitumor drugs such astriazenoimidazole (such as dacarbazine), mitomycin C, leinamycin, andthe like.

Examples of suitable DNA strand breakers include doxorubicin anddaunorubicin (which are also reversible DNA binders), otheranthracyclines, bleomycins, tirapazamine, enediyne antitumor antibioticssuch as neocarzinostatin, esperamicins, calicheamicins, dynemicin A,hedarcidin, C-1027, N1999A2, esperamicins, zinostatin, and the like.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. Treatment of cancer can be evidenced, for example,by a reduction in tumor size, a reduction in tumor burden, a reductionin clinical symptoms resulting from the cancer, increase in longevity,increase in tumor free survival time, and the like. Treating inembodiments, can include inhibiting the development or progression of acancer or metastatic cancer.

By the term “coadminister” is meant that each of the at least twocompounds be administered during a time frame wherein the respectiveperiods of biological activity overlap. Thus, the term includessequential as well as coextensive administration of two or more drugcompounds. The compounds can be administered simultaneously, separately(chronologically staggered), cyclically, or sequentially and in anyorder, such as before or after.

One skilled in the art will appreciate that suitable methods ofutilizing a compound and administering it to a human for the treatmentor prevention of disease states, in particular, cancer, which would beuseful in the method of the present invention, are available. Althoughmore than one route can be used to administer a particular compound, aparticular route can provide a more immediate and more effectivereaction than another route. Accordingly, the described methods aremerely exemplary and are in no way limiting.

The dose administered to a mammal, particularly, a human, in accordancewith the present invention should be sufficient to effect the desiredresponse. Such responses include reversal or prevention of the adverseeffects of the disease for which treatment is desired or to elicit thedesired benefit. One skilled in the art will recognize that dosage willdepend upon a variety of factors, including the age, condition, and bodyweight of the human, as well as the source, particular type of thedisease, and extent of the disease in the human. The size of the dosewill also be determined by the route, timing and frequency ofadministration as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular compound and the desired physiological effect. It will beappreciated by one of skill in the art that various conditions ordisease states may require prolonged treatment involving multipleadministrations.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 to about 300 mg of one or moreof the compounds described above per kg body weight of the animal ormammal.

The therapeutically effective amount of the compound or compoundsadministered can vary depending upon the desired effects and the factorsnoted above. Typically, dosages will be between 0.01 mg/kg and 250 mg/kgof the subject's body weight, and more typically between about 0.05mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, fromabout 5 to about 40 mg/kg or from about 10 to about 30 mg/kg of thesubject's body weight. Thus, unit dosage forms can be formulated basedupon the suitable ranges recited above and the subject's body weight.The term “unit dosage form” as used herein refers to a physicallydiscrete unit of therapeutic agent appropriate for the subject to betreated.

Alternatively, dosages are calculated based on body surface area andfrom about 1 mg/m² to about 200 mg/m², such as from about 5 mg/m² toabout 100 mg/m² will be administered to the subject per day. Inparticular embodiments, administration of the therapeutically effectiveamount of the compound or compounds involves administering to thesubject from about 5 mg/m² to about 50 mg/m², such as from about 10mg/m² to about 40 mg/m² per day. It is currently believed that a singledosage of the compound or compounds is suitable, however atherapeutically effective dosage can be supplied over an extended periodof time or in multiple doses per day. Thus, unit dosage forms also canbe calculated using a subject's body surface area based on the suitableranges recited above and the desired dosing schedule.

In accordance with other embodiments, the invention provides a method ofpotentiating or enhancing anticancer activity of radiation treatment,the method comprising coadministering to a patient in need thereof aneffective amount of a radiation treatment and a compound or salt of theinvention. The radiation treatment can be any suitable radiationtreatment used in the treatment of cancers.

The invention further provides a use of a compound or salt of theinvention in the manufacture of a medicament for treating or preventingcancer. The medicament typically is a pharmaceutical composition asdescribed herein.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

General Chemistry. Reagents and solvents used were commercial anhydrousgrade and used without further purification. Column chromatography wascarried out over silica gel (100-200 mesh). ¹H NMR spectra were recordedwith a Bruker 400 MHz spectrometer from solutions in CDCl₃ and DMSO-d6.Chemical shifts in ¹H NMR spectra are reported in parts per million(ppm, δ) downfield from the internal standard Me₄Si (TMS, δ=0 ppm).Chemical shifts in ¹³C NMR spectra are reported in parts per million(ppm, δ) calibrated from residual CHCl₃ (δ=77.0 ppm) signal and arereported using an APT pulse sequence displaying methyl and methine (CH₃and CH) signals as down and quaternary and methylene (C and CH₂) signalsas up. Molecular weight confirmation was performed using an Agilent 6224Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, SantaClara, Calif.). A 3 minute gradient from 5 to 100% Acetonitrile in water(0.03% formic acid) was used with a 5.1 minute run time at a flow rateof 0.4 mL/min. A Waters Atlantis T3 CIS column (1.8 micron, 2.1×50 mm)was used at a temperature of 25° C. Confirmation of molecular formulawas confirmed using electrospray ionization in the positive mode withthe Agilent Masshunter software (version B.02).

Example 1

This Example illustrates a procedure for the synthesis of2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile A, anintermediate in the synthesis of a compound in accordance with anembodiment of the invention.

A modified Voigt reaction/Knoevenagel condensation sequence was carriedout using the procedure described in Roth, H. J. et al., Arch. Pharmaz.1975, 308, 179-185. Benzoin (2.19 g, 10.3 mmol), benzylamine (1.66 g,15.5 mmol, 1.5 equiv.), and zinc chloride (0.10 g, 0.73 mmol, 0.07equiv.) were heated at reflux for 3 hours and the mixture was removedfrom the oil bath. To the still warm mixture was added malononitrile(1.35 g, 20.64 mmol, 2.0 equiv.) in DMF (3 mL). The reaction mixture wasallowed to cool to room temperature and stirred for 16 hrs, affordingthe crude pyrrole as a dark brown solid. The solid was partitionedbetween water and CH₂Cl₂ and the aqueous extracted with additionalCH₂Cl₂ (2×50 mL). The combined organics were dried with Na₂SO₄ and thesolvent removed in vacuo to afford the previously reported pyrroleproduct A as a light brown solid (1.67 g, 4.78 mmol, 46% yield), whichwas used without further purification. R_(f)=0.22 (20% EtOAc inhexanes); ¹H NMR δ 4.91 (s, 2H), 7.06-7.37 (complex, 15 HI); ¹³C NMR δ d(CH, CH₃) 125.8 (×2), 126.3, 127.9, 128.1, 128.2 (×2), 128.6 (×2), 128.7(×2), 129.2 (×2), 131.0; u (C, CH₂) 46.9, 117.5, 120.9, 125.6, 130.8,133.1, 136.0, 146.0, 162.5; IR 3329, 3228, 3031, 2195, 1663, 1556 cm⁻¹;HRMS calcd for C₂₄H₂₀N₃ [M+H⁺] 350.1657. found 350.1648.

Example 2

This Example illustrates a procedure for the synthesis of (E)-EthylN-(l-benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)formimidate B, anintermediate in the synthesis of a compound in accordance with anembodiment of the invention.

2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile A (1.07 g, 3.06mmol) and triethylorthoformate (4.54 g, 30.6 mmol, 10 equiv.) wereheated at 75° C. for 14 hrs and the excess triethylorthoformate wasremoved in vacuo. The residue was dissolved in a minimum of CH₂Cl₂,adsorbed onto celite, and chroomatographed on silica to afford theformimidate product B as a tan solid (0.80 g, 1.97 mmol, 64% yield).R_(j)=0.47 (20% EtOAc in hexanes); mp=154-156° C.; ¹H NMR δ 1.30 (t,J=7.2 Hz, 3H), 4.27 (dq, J=0.8, 8.2 Hz, 2H), 5.05 (s, 2H), 6.86 (dd,J=2.0, 8.0 Hz, 2H), 7.06 (dd, J=1.6, 8.0 Hz, 2H), 7.14-7.29 (complex,11H), 8.51 (s, 1H); ¹³C NMR δ d (CH, CH₃) 13.9, 126.4 (×2), 126.5,127.2, 128.1 (×2), 128.2, 128.4 (×4), 129.0 (×2), 131.2 (×2), 158.3; u(C, CH₂) 46.9, 63.2, 117.9, 123.1, 128.5, 130.8, 132.8, 137.6, 143.9; IR2208, 1627, 1605 cm⁻¹; HRMS calcd for C₂₇H₂₄N₃O [M+H⁺] 406.1919. found406.1915.

Example 3

This Example illustrates a synthesis of a compound in accordance with anembodiment of the invention,trans-4-(7-Benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)cyclohexanol2.

A solution of the formimidate B (40 mg, 0.099 mmol) andtrans-4-aminocyclohexanol hydrochloride (23 mg, 0.15 mmol, 1.5 equiv) inMeOH (1.5 mL) were heated in a reaction vial at 60° C. for 17 hrs thencooled to room temperature. Evaporation of the solvent and purificationof the residue by mass-directed preparative reverse-phase HPLC affordedthe pyrolopyrimidine product 2 as a tan solid (16 mg, 0.034 mmol, 34%yield). R_(f)=0.39 (1:1 acetone:CH₂Cl₂ with 1% Et₃N); mp=171-185° C.; ¹HNMR δ 1.66 (m, 4H), 2.13 (d, J=8.8 Hz, 4H), 3.70 (m, 1H), 5.14 (m, 1H),5.28 (s, 2H), 6.45 (br s, 1H), 6.95 (m, 2H), 7.04 (d, J=6.8 Hz, 2H),7.18-7.26 (complex, 11H), 7.80 (s, 1H); ¹³C NMR δ d (CH, CH₃) 52.0,69.7, 126.7 (×2), 126.9, 127.3, 128.0, 128.1 (×2), 128.3 (×2), 128.4(×2), 130.5 (×2), 131.0 (×2), 142.3; u (C, CH₂) 30.6, 34.7, 46.0, 102.9,118.1, 130.4, 133.2, 133.6, 137.7, 142.5, 155.1; IR 1625, 1604 cm⁻¹;HRMS calcd for C₃₁H₃₁N₄O [M+H⁺] 475.2498. found 475.2492.

Example 4

This Example illustrates a procedure for the synthesis of2-amino-1-phenethyl-4,5-diphenyl-1-H-pyrrole-3-carbonitrile C, anintermediate in the synthesis of a compound in accordance with anembodiment of the invention.

Benzoin (4.35 g, 20.5 mmol), phenethylamine (3.73 g, 30.7 mmol), andzinc chloride (0.10 g, 0.73 mmol, 0.07 equiv.) were heated at reflux for3 hours and the mixture was removed from the oil bath. To the still warmmixture malononitrile (2.71 g, 41.0 mmol, 2.0 equiv.) in DMF (3 mL) wasadded. The reaction mixture was allowed to cool to room temperature andstirred for 16 hrs. affording the crude pyrrole as a dark brown solid.The solid was partitioned between water and CH₂Cl₂ and the aqueousextracted with additional CH₂Cl₂ (2×50 mL). The combined organics weredried with Na₂SO₄ and the solvent removed in vacuo to afford pryrroleproduct C (3.50 g, 9.63 mmol, 47% yield) as a light brown solid.R_(f)=0.13 (20% EtOAc in hexanes); mp=144-149° C.; IR 3330, 2199, 1634,1601, 1502 cm⁻¹; HRMS calcd for C₂₅H₂₃N₃ [M+H⁺] 364.1814. found364.1827.

Example 5

This example demonstrates a high content assay for PNC detection.

The quantitative output for this assay is the reduction of PNCprevalence. The PNC can be detected in living cells by the expression ofa green fluorescent protein (GFP) tagged to the PNC localized protein,PTB. A PC3M cell line was used that stably expresses GFP-PTB to markPNCs. This method eliminates the need for immunofluorescent staining.Previous studies demonstrate that the fusion proteins behave similarlyto their endogenous counterparts: transient and stable over-expressionof the fusion protein did not have detectable adverse effects on cellmorphology or cell growth. After treatment, cells are fixed and thenuclei are counterstained with Hoechst 33342 dye; the cells are thenready for analysis.

The IN Cell Analyzer 1000 automated fluorescent imaging system (GEHealthcare, Piscataway, N.J.) was used for automated image acquisition.Images were acquired with a 20× objective using a 475/20 nm excitationfilter, a 535/50 nm HQ emission filter, a Q505LP dichroic filter, and anexposure time of 100 to 150 ms (adjusted to obtain a dynamic range of˜200 to 1750), with no camera binning. The instrument acquired images ofeach well in a 1536-well plate with a laser-based autofocus system. Toscore PNC prevalence in a high-content throughput, the Multi-TargetAnalysis (MTA) algorithm (GE Healthcare, Investigator v3.5) to identifyindividual cells and granules (PNCs) within these cells was used. Thenucleus was segmented via a region growing method (50 μm² minimum area)with light shading and noise removal to allow “touching” nuclei to beseparated. Granules in the nucleus (PNCs) were segmented using amultiscale top hat method, which measures granules of 1 to 2 μm in sizeand used a smart masking method to identify the boundaries of eachsegmented granule. The algorithm was optimized and validated usingpositive and negative controls (50 μM camptothecin and DMSO,respectively). In particular, the MTA algorithm allows for theidentification of multiple subcellular compartments and organelles (orgranules) within those compartments. In this instance, the algorithm'scapability to identify objects within the same color channel that onlydiffer in size or fluorescent intensity was utilized. Also, thealgorithm allows for building complex hierarchical classificationsystems, using output measures within the algorithm to filter and definesubpopulations. For this particular assay, PNC-positive cells werescored when 1 to 3 PNC granules were detected per nucleus. Cells thatcontained 0 granules were scored as PNC negative, and cells with >3granules were assumed to be false positives (very few cells have morethan 3 PNCs in one focus plane), and were also scored as PNC negative.The assay was conducted using the sequence set forth in Table 1.

TABLE 1 Sequence Value Parameter Description 1 Cells  5 μL 750-1000cells/well 2 Time  4 hrs Incubate at 37° C. and 5% CO₂ 3 Reagent 23 nL0.5 nM to 58 μM final concentrations (in titration) 4 Time 16 hrsIncubate at 37° C. and 5% CO₂ 5 Reagent  4 μL Fixation step with 6% EMgrade paraformaldehyde and 0.1% Triton X-100 6 Time 20 min RT incubation7 Wash  5 μL Liquid was aspirated and 5 μL of PBS was added 8 Wash  5 μLLiquid was aspirated and 5 μL of PBS was added 9 Reagent  5 μL Stainingwith PBS containing 1 μg/mL Hoechst 33342 10 Detector Fluorescence INCell 1000, 20 x objective

Example 6

This example demonstrates an adenosine triphosphate (ATP) quantitationassay.

This follow-up assay was conducted to measure the effect of compounds oncell health by measuring ATP levels (ATPLite™). ATPLite™ is an ATPmonitoring system based on firefly (Photinus pyralis) luciferase. Thelevel of ATP in a metabolically active cell is a general marker for itsviability. ATP levels are often reduced during necrosis or apoptosis. Inthis assay, the luciferase enzyme catalyzes the conversion of the addedsubstrate D-luciferin to oxyluciferin and light with ATP. Thus, theemitted light is proportional to the ATP concentration. For this assay,the highly metastatic PC3M reporter cell line stably expressing thePTB-GFP was provided by Professor Sui Huang of Northwestern University.The media and cell culture reagents were purchased from Invitrogen(Carlsbad, Calif.), ATPLite™ came from PerkinElmer. The assay wasconducted using the sequence set forth in Table 2.

TABLE 2 Sequence Value Parameter Description 1 Cells  5 μL 2000cells/well 2 Time  4 hrs Incubate at 37° C. and 5% CO₂ 3 Reagent 23 nL0.5 nM to 58 μM final concentrations (in titration) 4 Time 24 hrsIncubate at 37° C. and 5% CO₂ 5 Reagent  3 μL ATPLite 6 Time 20 min RTincubation 7 Centrifuge  1 min 1500 RPM 8 Detector Luminescence ViewLux

Example 7

This example demonstrates a tumor cell migration assay.

High-content tumor cell migration assays in 3-dimensional extracellularmatrices are powerful tools for modeling and understanding the biologyof this critical step in the process of metastasis. However, most of theavailable methods are not amenable to the throughput required by studiesof comparative pharmacology or small scale screening. For this reason,compounds were tested in BeliBrook Labs™ automated high-content tumorcell invasion assays. A standard screening-sized plate with an array ofembedded microchannels was designed and constructed from commonthermoplastics.

PC3M cells were tested for invasion through 3D fibrillar collagen in theluvo Single Microchannel Plate, in the presence of varying levels oftest compounds. Channels were prefilled with 820 nL of 3-dimensionaltype I collagen at 1 mg/mL, through the input port. Following gelation,2,000 PC3M cells were seeded into the output port using growth media(RPMI+10% FBS with antibiotics) in a volume of 5 μL. Cells wereincubated in a 37° C. incubator inside a humidified container to controlevaporation (Bioassay dish, Corning). Media, including test compounds,was changed daily for 5 days. At the end of the assay, cells were fixedand stained with Hoechst 33342, then imaged with 4× objective underepifluorescence. Under these conditions, cells across the 140 μm heightrange of the microchannel can be reliably identified. Test compoundswere serially diluted by a factor of 3 to produce 10 concentrationsranging from 50 or 100 μM to 2.5 nM. All assays were conducted in thepresence of 0.1% DMSO. Four replicates were performed for all testconcentrations. Each plate had 4 dose response curves, as well as 16channels with no compound and 16 channels with 50 μM blebbistatin(positive control). Analysis was done by automatically cropping eachimage at the right edge of the channel and counting cells via the ‘countnuclei’ function on Metamorph (Molecular Devices). Non-linear regressionanalysis was performed with GraphPad Prism. The results are set forth inTable 3.

TABLE 3 Proliferation Invasion output PNC cell # port IC₅₀ Compound AC₅₀(μM) IC₅₀ (μM) (μM) Comments 2 0.40 3.16 3.98 embodiment Controlinactive 79.43 19.95 Negative control 1 0.09 3.16 5.01 embodiment

Example 8

This example demonstrates the effect of an embodiment of the inventionon colony formation of PC3M cells.

Compound 2 was tested for its ability to affect anchorage independentgrowth in a soft agar assay, a stringent method to detect malignanttransformation of cells in vitro. Compound 2 demonstrated a dosedependent reduction in the number of colonies after 14 days at very lowconcentrations (3.8, 18.6 nM), with no impact on cell viability. Thus,the compound exhibits potent inhibition of anchorage independent growthin PC3M cells. The left hand side of FIG. 1 illustrates a histogramrepresenting the number and size of PC3M soft agar colonies after 14days of treatment with compound 2 at two different concentrations. Aclear reduction of the number of colonies of PC3M cells was observed,particularly at a compound concentration of 18.6 nM. The right hand sideof FIG. 1 illustrates two representative images taken at two differentregions of the soft agar medium after treatment with DMSO (vehicle) (toprow), compound 2 at 3.8 nM (middle row), and compound 2 at 18.6 nM(bottom row).

Example 9

This example demonstrates biological activities of embodiments of theinvention. The high content assay for PNC detection as described inExample 5 was used to provide the PNC AC₅₀ results. The ATP quantitationassay as described in Example 6 was used to provide the ATP AC₅₀results. The results are set forth in Table 4.

TABLE 4 PNC ATP Compound Structure IC₅₀(μM) IC₅₀(μM) 56

0.009 19.182 2

0.024 19.182 1

0.030 152.369 3

0.047 9.614 57

0.059 96.138 4

0.118 76.365 61

0.118 24.149 59

0.118 24.149 60

0.118 24.149 58

0.118 24.149 9

0.148 19.182 63

0.148 19.182 62

0.148 24.149 8

0.148 24.149 5

0.187 19.182 64

0.187 38.273 13

0.235 15.237 65

0.235 19.182 67

0.296 24.149 66

0.296 76.365 68

0.296 38.273 15

0.372 24.149 71

0.372 121.031 70

0.372 24.149 69

0.372 19.182 72

0.469 24.149 20

0.469 96.138 16

0.590 38.273 73

0.590 24.149 7

0.628 20.434 74

0.743 24.149 19

0.935 24.149 38

0.935 96.138 76

0.935 48.183 75

0.935 24.149 14

1.177 24.149 27

1.177 24.149 17

1.177 30.402 77

1.177 96.138 8

1.254 20.434 37

1.254 25.725 34

1.254 20.434 6

1.482 48.183 78

1.482 30.402 25

1.866 60.659 24

1.866 24.149 81

1.866 76.365 80

1.866 21.523 79

1.866 382.734 36

1.987 25.725 82

2.349 30.402 31

2.957 24.149 83

2.957 24.149 33

2.957 48.183 85

2.957 24.149 84

2.957 24.149 26

3.722 60.659 86

3.722 24.149 11

3.965 64.618 23

4.686 121.031 39

4.686 48.183 48

4.686 30.402 88

4.686 30.402 87

4.686 60.659

5.899 38.273 30

5.899 24.149 40

5.899 38.273 90

5.899 76.365 89

5.899 24.149 91

5.899 30.402 21

7.427 24.149 22

7.427 24.149 97

7.427 96.138 93

7.427 60.659 45

9.350 24.149 94

9.350 24.149 43

9.960 20.434 96

11.770 30.402 95

11.770 24.149 99

14.818 inactive 46

14.818 24.149 98

14.818 121.031 101

14.818 24.149 97

14.818 48.183 100

14.818 48.183 102

15.785 25.725 141

15.785 20.434 32

18.655 24.149 103

18.655 24.149 29

23.485 27.096 104

23.485 30.402 106

23.485 30.402 105

23.485 304.016 107

25.018 32.386 108

29.566 38.273 53

29.566 24.149 37

39.650 81.349 110

46.859 30.402 109

46.859 96.138 111

93.496 121.031 137

inactive inactive 50

inactive 32.386 116

inactive inactive 10

inactive 96.138 114

inactive inactive 140

inactive inactive 118

inactive 121.031 125

inactive 30.402 10

inactive 48.183 120

inactive 241.489 139

inactive inactive 128

inactive inactive 130

inactive 24.149 136

inactive inactive 131

inactive 121.031 119

inactive 121.031 117

inactive 96.138 122

inactive inactive 115

inactive 121.031 127

inactive 96.138 124

inactive inactive 138

inactive 60.659 121

inactive 121.031 12

inactive 24.149 18

inactive 76.365 112

inactive 60.659 132

inactive inactive 126

inactive 96.138 129

inactive inactive 135

inactive 30.402 134

inactive 60.659 123

inactive 24.149 113

inactive 30.402 35

Inactive 19.87 44

15.79 19.87

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The terms “comprising,” “having,” “including,” and “containing” are tobe construed as open-ended terms (that is meaning “including, but notlimited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (such as, “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-46. (canceled)
 47. A method for treating cancer in a mammal,comprising administering to a mammal in need thereof a compound offormula (I):

wherein R¹ is selected from the group consisting of alkyl, hydroxyalkyl,thioalkyl, alkoxyalkyl, alkylthioalkyl, cycloalkyl, hydroxycycloalkyl,hydroxycycloalkylalkyl, thiocycloalkyl, alkoxycycloalkyl,alkylthiocycloalkyl, dialkylaminoalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, arylalkyl, arylalkylpiperidin-4-yl, arylpiperazinylalkyl,and heteroarylalkyl, R² is phenyl, optionally substituted with one ormore substituents selected from the group consisting of halo, alkyl,hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl,alkylthiocarbonyl, amino, alkylamino, dialkylamino, and alkylcarbonyl,R³ is phenyl, optionally substituted with one or more substituentsselected from the group consisting of halo, alkyl, hydroxyalkyl,thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl,amino, alkylamino, dialkylamino, and alkylcarbonyl, R⁴ is selected fromthe group consisting of alkyl, cycloalkyl, cycloalkylalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl, or a pharmaceuticallyacceptable salt thereof, wherein R¹ and R⁴ are optionally substituted onthe aryl and/or alkyl portion with one or more substituents selectedfrom the group consisting of halo, alkyl, hydroxyalkyl, thioalkyl,alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl, amino,alkylamino, dialkylamino, aminosulfonyl, hydroxyl, perfluoroalkoxy,alkylenedioxy, and alkylcarbonyl, wherein the cancer is a metastaticcancer selected from the group consisting of breast cancer, ovariancancer, colorectal cancer, brain cancer, prostate cancer, and pancreaticcancer.
 48. The method of claim 47, wherein R¹ is a 5 or 6-memberedheterocyclyl group having at least one hetero atom selected from thegroup consisting of O, N, and S; a hydroxy C₁-C₇ cycloalkyl group; ahydroxy C₁-C₆ alkyl group; a N,N-di(C₁-C₆ alkyl)amino C₁-C₆ alkyl group;a C₁-C₆ alkoxy C₁-C₆ alkyl group; a heteroaryl C₁-C₆ alkyl group; aheterocyclyl C₁-C₆ alkyl group; phenyl C₁-C₆ alkyl group where thephenyl ring is substituted with one or more C₁-C₆ alkoxy groups;N-benzyl piperazinyl; N-phenyl piperazinylalkyl; a phenyl C₁-C₆ alkylgroup where the alkyl is substituted with a hydroxy group; or a 5 or 6membered heteroarylamino C₁-C₆ alkyl group wherein the heteroaryl grouphas at least one hetero atom selected from the group consisting of O, N,and S.
 49. The method of claim 47, wherein R¹ is selected from the groupconsisting of the following:


50. The method of claim 49, wherein R² is phenyl, R³ is phenyl, R⁴ isbenzyl, and R¹ is selected from the group consisting of the following:


51. The method of claim 47, wherein R⁴ is 4-methoxybenzyl, R² is phenyl,R³ is phenyl, and R¹ is selected from the group consisting of thefollowing:


52. The method of claim 47, wherein R⁴ is phenylethyl, R² is phenyl, R³is phenyl, and R¹ is selected from the group consisting of thefollowing:


53. The method of claim 47, wherein R⁴ is selected from4-aminosulfonylbenzyl, 4-trifluoromethoxybenzyl, 4-methoxybenzyl, andcyclopropylmethyl and wherein R¹ is selected from the following:


54. The method of claim 47, wherein R⁴ is heteroaryl C₁-C₆ alkyl. 55.The method of claim 47, wherein R² is phenyl, R³ is phenyl, R⁴ is

and R¹ is selected from the group consisting of the following:


56. The method of claim 47, wherein the cancer is pancreatic cancer. 57.The method of claim 47, further comprising administering to the mammal achemotherapeutic agent or subjecting the mammal to a radiationtreatment.
 58. The method of claim 47, wherein the treating results inthe disrupting a perinucleolar compartment in a cell in the mammal,reducing the prevalence of perinucleolar compartment in a cell in themammal, reducing ATP levels produced by metastatic cancer cells in themammal, reducing the colony formation of cancer cells in the mammal, orreducing the migration of cancer cells in the mammal.
 59. A compound offormula (I):

wherein R¹ is selected from the group consisting of alkyl, thioalkyl,alkoxyalkyl, alkylthioalkyl, cycloalkyl, hydroxycycloalkyl,hydroxycycloalkylalkyl, thiocycloalkyl, alkoxycycloalkyl,alkylthiocycloalkyl, dialkylaminoalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, arylalkyl, arylalkylpiperidin-4-yl, andarylpiperazinylalkyl, R² is phenyl, optionally substituted with one ormore substituents selected from the group consisting of halo, alkyl,hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl,alkylthiocarbonyl, amino, alkylamino, dialkylamino, and alkylcarbonyl,R³ is phenyl, optionally substituted with one or more substituentsselected from the group consisting of halo, alkyl, hydroxyalkyl,thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl,amino, alkylamino, dialkylamino, and alkylcarbonyl, R⁴ isheteroarylalkyl, or a pharmaceutically acceptable salt thereof, whereinR¹ and R⁴ are optionally substituted on the aryl and/or alkyl portionwith one or more substituents selected from the group consisting ofhalo, alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl,alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino,aminosulfonyl, hydroxyl, perfluoroalkoxy, alkylenedioxy, andalkylcarbonyl.
 60. The compound or salt of claim 59, wherein R² isphenyl, R³ is phenyl, R⁴ is

and R¹ is selected from the group consisting of the following:


61. A pharmaceutical composition comprising a compound or salt of claim47 and a pharmaceutically acceptable carrier.